Fig. 2

Interaction and colocalization of NS1 and TRAF3 in mammalian cells. A HEK293T cells were transfected with His-MAVS together with different NS1-Myc/His constructs. Cell lysates were subjected to the immunoprecipitation (IP)- Western blot (WB) analysis. B Like panel A, the IP-WB analysis was conducted to examine the interaction of NS1-Myc/His and Flag-TRAF3 or Flag-TRAF6 in HEK293 T cells. C, D Similarly, the IP-WB analysis was used to map the domain of NS1 critical for binding TRAF3 by transfection of Flag-TRAF3 with NS1-Myc/His or different NS1 truncated mutants as indicated. E HEK293T cells transfected with Flag-TRAF3 or Flag-TRAF6 were infected with IAV (PR8, 3 MOI) to examine the interaction of NS1and TRAF3 under virus infection by the IP-WB analysis. F Schematic structures of TRAF3 and its truncated mutants with an N-terminal Flag-tag. G A similar IP-WB analysis was conducted to determine the domain of TRAF3 for binding NS1 by transfection of NS1-Myc/His with Flag-TRAF3 or its truncated mutants as indicated. * means the non-specific band. H HEK293T cells were transfected with Flag-TRAF3 alone or in combination with GFP-tagged MAVS, NS1-Myc/His, or NS1-ED-Myc/His as indicated. Then, the cells were harvested for the IP- WB analysis to assess the effect of NS1 and NS1-ED on the complex formation between MAVS and TRAF3. I Confocal microscopy was performed to examine the colocalization of NS1 and TRAF3 in mammalian cells. HEK293 cells were transfected with NS1-Myc/His together with Flag-TRAF3, and the cells were then subjected to the immunofluorescence analysis using anti-Myc and anti-TRAF3 antibodies. J Like panel I, HEK293 cells transfected with Flag-RIG-I were left untreated or infected with IAV (PR8, 5 MOI) for 16 h. Then the cells were subjected to confocal microscopy using anti-NS1 and anti-TRAF3 antibodies